Fig. 3

PLK1 phosphorylates RhoGDI1 at Thr7/91 residue in vitro and in vivo. a Purified GST-RhoGDI1 was subjected to in vitro kinase assay with( +) or without(-) active His-PLK1. The assay products were immunoblotted with the indicated antibodies. b Flag-RhoGDI1 was transfected with or without HA-PLK1 into HeLa cells. Cell lysates were immunoprecipitated with Flag antibody. Immunoprecipitates and total lysates were immunoblotted with the indicated antibodies. c HeLa cells were co-transfected Flag-RhoGDI1 with the control siRNA or two PLK1 siRNAs. Cell lysates were analyzed by Immunoprecipitation and immunoblotting using the indicated antibodies. d Purified His-RhoGDI1 wt and threonine-to-alanine-substituted mutants (T7A, T91A and T7/91A) were subjected to in vitro kinase assay with (+) or without (−) active His-PLK1. The assay products were immunoblotted with the indicated antibodies. e HeLa cells were transfected with Flag-RhoGDI1 wt or T7/91A and then treated with 200 nM of BI6727 (PLK1 inhibitor) for 4 h. Cell lysates were immunoprecipitated with Flag antibody. Immunoprecipitates and total lysates were immunoblotted with the indicated antibodies